Methods for performing multiplexed real-time PCR

ABSTRACT

The present invention describes methods for performing higher multiplexed real-time PCR for detection and quantitation of target nucleic acids using tagged hydrolysis probes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/705,821 filed Sep. 15, 2017, which claims the benefit ofpriority to U.S. Provisional Application No. 62/395,325, filed on Sep.15, 2016, U.S. Provisional Application No. 62/435,595, filed on Dec. 16,2016, and U.S. Provisional Application No. 62/536,871, filed on Jul. 25,2017, each of which is hereby incorporated in its entirety by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “33677_US2.txt”, having a size in bytes of 2 kb, andcreated on Aug. 14, 2017. The information contained in this electronicfile is hereby incorporated by reference in its entirety pursuant to 37CFR § 1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to methods for polymerase chain reaction(PCR) particularly to methods for performing multiplexed real-time PCR.

BACKGROUND OF THE INVENTION

The polymerase chain reaction (PCR) has become a ubiquitous tool ofbiomedical research, disease monitoring and diagnostics. Amplificationof nucleic acid sequences by PCR is described in U.S. Pat. Nos.4,683,195, 4,683,202, and 4,965,188. PCR is now well known in the artand has been described extensively in the scientific literature. See PCRApplications, ((1999) Innis et al., eds., Academic Press, San Diego),PCR Strategies, ((1995) Innis et al., eds., Academic Press, San Diego);PCR Protocols, ((1990) Innis et al., eds., Academic Press, San Diego),and PCR Technology, ((1989) Erlich, ed., Stockton Press, New York). A“real-time” PCR assay is able to simultaneously amplify and detect andquantify the starting amount of the target sequence. The basic TaqManreal-time PCR assay using the 5′-to-3′ nuclease activity of the DNApolymerase is described in Holland et al., (1991) Proc. Natl. Acad. Sci.88:7276-7280 and U.S. Pat. No. 5,210,015. The real-time PCR without thenuclease activity (a nuclease-free assay) has been described in a U.S.application Ser. No. 12/330,694 filed on Dec. 9, 2008. The use offluorescent probes in real-time PCR is described in U.S. Pat. No.5,538,848.

A typical real-time PCR protocol with fluorescent probes involves theuse of a labeled probe, specific for each target sequence. The probe ispreferably labeled with one or more fluorescent moieties, which absorband emit light at specific wavelengths. Upon hybridizing to the targetsequence or its amplicon, the probe exhibits a detectable change influorescent emission as a result of probe hybridization or hydrolysis.

The major challenge of the real-time assay however remains the abilityto analyze numerous targets in a single tube. In virtually every fieldof medicine and diagnostics, the number of loci of interest increasesrapidly. For example, multiple loci must be analyzed in forensic DNAprofiling, pathogenic microorganism detection, multi-locus geneticdisease screening and multi-gene expression studies, to name a few.

With the majority of current methods, the ability to multiplex an assayis limited by the detection instruments. Specifically, the use ofmultiple probes in the same reaction requires the use of distinctfluorescent labels. To simultaneously detect multiple probes, aninstrument must be able to discriminate among the light signals emittedby each probe. The majority of current technologies on the market do notpermit detection of more than four to seven separate wavelengths in thesame reaction vessel. Therefore, using one uniquely-labeled probe pertarget, no more than four to seven separate targets can be detected inthe same vessel. In practice, at least one target is usually a controlnucleic acid. Accordingly, in practice, no more than three to sixexperimental targets can be detected in the same tube. The use offluorescent dyes is also limited due to the spectral width where onlyabout six or seven dyes can be fit within the visible spectrum withoutsignificant overlap interference. Thus the ability to multiplex an assaywill not keep pace with the clinical needs, unless radical changes inthe amplification and detection strategy are made.

An additional ability to multiplex a real-time amplification reaction isprovided by a post-PCR melting assay. See U.S. patent application Ser.No. 11/474,071, filed on Jun. 23, 2006. In a melting assay, theamplified nucleic acid is identified by its unique melting profile. Amelting assay involves determining the melting temperature (meltingpoint) of a double-stranded target, or a duplex between the labeledprobe and the target. As described in U.S. Pat. No. 5,871,908, todetermine melting temperature using a fluorescently labeled probe, aduplex between the target nucleic acid and the probe is gradually heated(or cooled) in a controlled temperature program. The dissociation of theduplex changes the distance between interacting fluorophores or betweena fluorophore and a quencher. The interacting fluorophores may beconjugated to separate probe molecules, as described in U.S. Pat. No.6,174,670. Alternatively, one fluorophore may be conjugated to a probe,while the other fluorophore may be intercalated into a nucleic acidduplex, as described in U.S. Pat. No. 5,871,908. As yet anotheralternative, the fluorophores may be conjugated to a single probeoligonucleotide. Upon the melting of the duplex, the fluorescence isquenched as the fluorophore to the quencher are brought together in thenow single-stranded probe.

The melting of the nucleic acid duplex is monitored by measuring theassociated change in fluorescence. The change in fluorescence may berepresented on a graph referred to as “melting profile.” Becausedifferent probe-target duplexes may be designed to melt (or reanneal) atdifferent temperatures, each probe will generate a unique meltingprofile. Properly designed probes would have melting temperatures thatare clearly distinguishable from those of the other probes in the sameassay. Many existing software tools enable one to design probes for asame-tube multiplex assay with these goals in mind. For example, VisualOMP™ software (DNA Software, Inc., Ann Arbor, Mich.) enables one todetermine melting temperatures of nucleic acid duplexes under variousreaction conditions.

The method of multiplex PCR using color detection and subsequentpost-amplification melting assay is described in U.S. Pat. No.6,472,156. The number of targets detectable by such a method is aproduct of the number of detectable wavelengths and the number ofdistinguishable melting profiles. Therefore adding a melting assay tocolor detection was a step forward in the ability to detect multipletargets.

The post-amplification melting assay is most commonly used forqualitative purposes, i.e. to identify target nucleic acids, see U.S.Pat. Nos. 6,174,670, 6,427,156 and 5,871,908. It is known to obtain amelting peak by differentiating the melting curve function. Ririe et al.(“Product differentiation by analysis of DNA melting curves during thepolymerase chain reaction,” (1997) Anal. Biochem. 245:154-160) observedthat differentiation helps resolve melting curves generated by mixturesof products. After differentiation, the melting peaks generated by eachcomponent of the mixture become easily distinguishable. It was alsopreviously known that the post-amplification melting signal, i.e.melting peak, is higher in proportion to the amount of the nucleic acidin the sample. For example, U.S. Pat. No. 6,245,514 teaches apost-amplification melt assay using a duplex-intercalating dye, togenerate a derivative melting peak, and then, using proprietarysoftware, to integrate the peak. The integration provides informationabout the efficiency of amplification and relative amount of theamplified nucleic acid.

In practice, it would be desirable to move beyond a qualitative assayand be able to quantify multiple targets in the same sample. See e.g.Sparano et al. “Development of the 21-gene assay and its application inclinical practice and clinical trials,” J. Clin. Oncol. (2008)26(5):721-728. The ability to quantify the amount of target is useful inclinical applications, such as determination of viral load in apatient's serum, measuring the level of expression of a gene in responseto drug therapy or determining the molecular signature of a tumor topredict its response to therapy.

In a real-time PCR assay, the signal generated by the labeled probe canbe used to estimate the amount of input target nucleic acid. The greaterthe input, the earlier the fluorescence signal crosses a predeterminedthreshold value (Ct). Therefore one can determine relative or absoluteamounts of the target nucleic acid by comparing the samples to eachother or to a control sample with known amount of nucleic acid. However,the existing methods are limited in their ability to simultaneouslyquantify multiple targets. As with the qualitative detection of multipletargets, the limiting factor is the availability ofspectrally-resolvable fluorophores. As explained above, state-of-the-artfluorescent label technology is not able to obtain distinct signals frommore than six or seven separate fluorescently labeled probes in the sametube. Therefore a radically different experimental approach is needed topermit amplification and detection of numerous nucleic acid targetsduring real-time PCR.

Many methods for detection of target nucleic acids are known. Currentlyavailable homogeneous assays for nucleic acid detection include theTaqMan®, Ampliflour®, dye-binding, allele-selective kinetic PCR andScorpion® primer assays. These assay procedures are not readilymultiplexed due to the requirement for a different dye for each targetnucleic acid to be detected, and thus are limited in their potential forimprovement. To overcome such limitations, several recent studies havedisclosed the use of oligonucleotide probes containing a cleavable “tag”portion which can be readily separated and detected (e.g. see Chenna etal, U.S. Patent Application Publication No. 2005/0053939; Van Den Boom,U.S. Pat. No. 8,133,701). More recently, improved methods to performmultiplexed nucleic acid target identification by using structure basedoligonucleotide probe cleavage have been described in U.S. PatentApplication Publication No. 2014/0272955, U.S. 2015/0176075, and U.S.2015/0376681, all incorporated by reference herein. Further methods todetect target nucleic acid sequence from DNA or a mixture of nucleicacid by the use of a combination of “Probing and TaggingOligonucleotide” (PTO) and “Capturing and Templating Oligonucleotide”(CTO) in a so-called PTO Cleavage and Extension assay have beendescribed by Chun et al. in U.S. Pat. No. 8,809,239. However the needstill exists for an accurate method to perform high throughput multiplexdetection of target nucleic acids.

SUMMARY OF THE INVENTION

The present invention provides for novel methods for nucleic acidsequence detection, particularly detection of multiple target nucleicacids using a real-time PCR assay. The methods are performed by the useof novel oligonucleotide probes having two unique features, anon-complementary tag portion and a quenching molecule.

Therefore in one aspect, the invention provides for a method foramplification and detection of a target nucleic acid in a samplecomprising the steps of: (a) contacting the sample containing the targetnucleic acid in a single reaction vessel with (i) one pair ofoligonucleotide primers, each oligonucleotide primer capable ofhybridizing to opposite strands of a subsequence of the target nucleicacid; (ii) an oligonucleotide probe that comprises an annealing portionand a tag portion, wherein the tag portion comprises a nucleotidesequence non-complementary to the target nucleic acid sequence or anon-nucleotide molecule, wherein the annealing portion comprises anucleotide sequence at least partially complementary to the targetnucleic acid sequence and hybridizes to a region of the subsequence ofthe target nucleic acid that is bounded by the pair of oligonucleotideprimers, wherein the probe further comprises an interactive dual labelcomprising a reporter moiety located on the tag portion or on theannealing portion and a first quencher moiety located on the annealingportion and wherein the reporter moiety is separated from the firstquencher moiety by a nuclease susceptible cleavage site; and wherein thetag portion is reversibly bound in a temperature-dependent manner to aquenching molecule that comprises or is associated with one or morequencher moieties capable of quenching the reporter moiety when thequenching molecule is bound to the tag portion; (b) amplifying thetarget nucleic acid by PCR using a nucleic acid polymerase having 5′ to3′ nuclease activity such that during an extension step of each PCRcycle, the nuclease activity of the polymerase allows cleavage andseparation of the reporter moiety from the first quenching moiety on theannealing portion of the probe; (c) measuring a suppressed signal fromthe reporter moiety at a first temperature at which the quenchingmolecule is bound to the tag portion; (d) increasing temperature to asecond temperature at which the quenching molecule is not bound to thetag portion; (e) measuring a temperature corrected signal from thereporter moiety at the second temperature; (f) obtaining a calculatedsignal value by subtracting the suppressed signal detected at the firsttemperature from the temperature corrected signal detected at the secondtemperature; (g) repeating steps (b) through (f) through multiple PCRcycles; (h) measuring the calculated signal values from the multiple PCRcycles to detect the presence of the target nucleic acid.

In one embodiment, the tag portion comprises a modification such that itis not capable of being extended by a nucleic acid polymerase. In oneembodiment, the reporter moiety is on the tag portion of theoligonucleotide probe. In another embodiment, the reporter moiety islocated on the annealing portion of the oligonucleotide probe and isable to interact in a temperature-dependent manner to the quenchingmolecule that comprises the second quencher moiety. In one embodiment,the tag portion comprises a nucleotide sequence non-complementary to thetarget nucleic acid sequence and the quenching molecule is anoligonucleotide comprising a nucleotide sequence at least partiallycomplementary to the tag portion of the oligonucleotide probe and bindsto the tag portion by hybridization. In another embodiment, the tagportion of the oligonucleotide probe or the quenching molecule or boththe tag portion and the quenching molecule contain one or morenucleotide modifications. In yet another embodiment, the one or morenucleotide modifications is selected from the group consisting of LockedNucleic Acid (LNA), Peptide Nucleic Acid (PNA), Bridged Nucleic Acid(BNA), 2′-O alkyl substitution, L-enantiomeric nucleotide, orcombinations theoreof. In one embodiment, the reporter moiety is afluorescent dye and the quencher moiety quenches a detectable signalfrom the fluorescent dye.

In another aspect, the invention provides for a method for detecting twoor more target nucleic acid sequences in a sample comprising the stepsof: (a) contacting the sample suspected of containing the two or moretarget nucleic acid sequences in a single reaction vessel with (i) afirst pair of oligonucleotide primers with nucleotide sequences that arecomplementary to each strand of a first target nucleic acid sequence,and a second pair of oligonucleotide primers with nucleotide sequencesthat are complementary to each strand of a second target nucleic acidsequence; (ii) a first oligonucleotide probe comprising a nucleotidesequence at least partially complementary to the first target nucleicacid sequence and anneals within the first target nucleic acid sequencebounded by the first pair of oligonucleotide primers, wherein the firstoligonucleotide probe comprises a fluorescent moiety capable ofgenerating a detectable signal and a first quencher moiety capable ofquenching the detectable signal generated by the fluorescent moiety,wherein the fluorescent moiety is separated the first quencher moiety bya nuclease susceptible cleavage site; (iii) a second oligonucleotideprobe comprising two distinct portions, an annealing portion comprisinga nucleotide sequence at least partially complementary to the secondtarget nucleic acid sequence and anneals within the second targetnucleic acid sequence bounded by the second pair of oligonucleotideprimers, wherein the annealing portion comprises a second quenchermoiety; and a tag portion attached to the 5′ terminus or to the 3′terminus of the annealing portion or attached via a linker to a regionof the annealing portion and comprising a nucleotide sequence that isnon-complementary to the two or more target nucleic acid sequences,wherein the tag portion comprises a fluorescent moiety that is identicalto the fluorescent moiety on the first oligonucleotide probe and whosedetectable signal is capable of being quenched by the second quenchermoiety on the annealing portion, wherein the fluorescent moiety isseparated from the second quenching moiety by a nuclease susceptiblecleavage site; (iv) a quenching oligonucleotide comprising a nucleotidesequence at least partially complementary to the tag portion of thesecond oligonucleotide probe and hybridizes to the tag portion to form aduplex, wherein the quenching oligonucleotide comprises a third quenchermoiety which quenches the detectable signal generated by the fluorescentmoiety on the tag portion when the quenching oligonucleotide ishybridized to the tag portion; (b) amplifying the first and secondtarget nucleic acid sequences by polymerase chain reaction (PCR) using anucleic acid polymerase having 5′ to 3′ nuclease activity such thatduring an extension step of each PCR cycle, the 5′ to 3′ nucleaseactivity of the nucleic acid polymerase allows cleavage and separationof the fluorescent moiety from the first quenching moiety on the firstoligonucleotide probe, and cleavage and separation of the fluorescentmoiety on the tag portion from the second quenching moiety on theannealing portion of the second oligonucleotide probe, wherein at theextension step the quenching oligonucleotide remains hybridized to thetag portion; (c) measuring a fluorescent signal at a first temperatureat which the quenching oligonucleotide is hybridized to the tag portionfrom the second oligonucleotide probe; (d) increasing temperature to asecond temperature, which is higher than the first temperature, at whichthe quenching oligonucleotide is not hybridized to the tag portion fromthe second oligonucleotide probe; (e) measuring a fluorescent signal atthe second temperature; (f) obtaining a calculated signal value bysubtracting the fluorescent signal detected at the first temperaturefrom the fluorescent signal detected at the second temperature; (g)repeating steps (b) through (f) in multiple PCR cycles to producedesired quantity of amplification products from the first and secondtarget nucleic acid sequences; (h) determining the presence of the firsttarget nucleic acid sequence from the fluorescent signals detected atthe first temperature from the multiple PCR cycles and the presence ofthe second target nucleic acid sequence from the calculated signalvalues from the multiple PCR cycles.

In one embodiment, the tag portion comprises a modification such that itis not capable of being extended by a nucleic acid polymerase. Inanother embodiment, the tag portion is attached to the 5′ terminus ofthe annealing portion. In yet another embodiment, the tag portion isattached to the 3′ terminus of the annealing portion. In yet anotherembodiment, the tag portion is attached via a linker to a region of theannealing portion. In another embodiment, the tag portion of the secondoligonucleotide probe or the quenching oligonucleotide or both the tagportion and the quenching oligonucleotide contain one or more nucleotidemodifications. In yet another embodiment, the one or more nucleotidemodifications is selected from the group consisting of Locked NucleicAcid (LNA), Peptide Nucleic Acid (PNA), Bridged Nucleic Acid (BNA), 2′-Oalkyl substitution, L-enantiomeric nucleotide, or combinations theoreof.

In yet another aspect, the invention provides for a method for detectingtwo or more target nucleic acid sequences in a sample comprising thesteps of: (a) contacting the sample suspected of containing the two ormore target nucleic acid sequences in a single reaction vessel with (i)a first pair of oligonucleotide primers with sequences that arecomplementary to each strand of a first target nucleic acid sequence,and a second pair of oligonucleotide primers with sequences that arecomplementary to each strand of a second target nucleic acid sequence;(ii) a first oligonucleotide probe comprising two distinct portions, afirst annealing portion comprising a sequence at least partiallycomplementary to the first target nucleic acid sequence and annealswithin the first target nucleic acid sequence bounded by the first pairof oligonucleotide primers, wherein the first annealing portioncomprises a first quencher moiety; and a first tag portion attached tothe 5′ terminus or to the 3′ terminus of the first annealing portion orattached via a linker to a region of the first annealing portion andcomprising a nucleotide sequence that is non-complementary to the two ormore target nucleic acid sequences, wherein the first tag portioncomprises a fluorescent moiety whose detectable signal is capable ofbeing quenched by the first quencher moiety on the first annealingportion, wherein the fluorescent moiety is separated from the firstquenching moiety by a nuclease susceptible cleavage site; (iii) a firstquenching oligonucleotide comprising a sequence at least partiallycomplementary to the first tag portion of the first oligonucleotideprobe and hybridizes to the first tag portion to form a duplex, whereinthe first quenching oligonucleotide comprises a second quenching moietywhich quenches the detectable signal generated by the fluorescent moietyon the first tag portion when the first quenching oligonucleotide ishybridized to the first tag portion; (iv) a second oligonucleotide probecomprising two distinct portions, a second annealing portion comprisinga sequence at least partially complementary to the second target nucleicacid sequence and anneals within the second target nucleic acid sequencebounded by the second pair of oligonucleotide primers, wherein thesecond annealing portion comprises a third quencher moiety; and a secondtag portion attached to the 5′ terminus or to the 3′ terminus of thesecond annealing portion or attached via a linker to a region of thesecond annealing portion and comprising a nucleotide sequence that isnon-complementary to the two or more target nucleic acid sequences, andhas a different nucleic sequence or different nucleotide modificationscompared to the nucleotide sequence of the first tag portion of thefirst oligonucleotide probe, wherein the second tag portion comprises afluorescent moiety that is identical to the fluorescent moiety on thefirst oligonucleotide probe and whose detectable signal is capable ofbeing quenched by the third quencher moiety on the second annealingportion, wherein the fluorescent moiety is separated from the thirdquenching moiety by a nuclease susceptible cleavage site; (v) a secondquenching oligonucleotide comprising a sequence at least partiallycomplementary to the second tag portion of the second oligonucleotideprobe and hybridizes to the second tag portion to form a duplex, whereinthe second quenching oligonucleotide comprises a fourth quenching moietywhich quenches the detectable signal generated by the fluorescent moietyon the second tag portion when the second quenching oligonucleotide ishybridized to the second tag portion; wherein the duplex between thesecond quenching oligonucleotide and the second tag portion of thesecond oligonucleotide probe has a higher melting temperature (Tm) valuethan the duplex between the first quenching oligonucleotide and thefirst tag portion of the first oligonucleotide probe; (b) amplifying thefirst and second target nucleic acid sequences by polymerase chainreaction (PCR) using a nucleic acid polymerase having 5′ to 3′ nucleaseactivity such that during an extension step of each PCR cycle, the 5′ to3′ nuclease activity of the nucleic acid polymerase allows (i) cleavageand separation of the fluorescent moiety on the first tag portion fromthe first quenching moiety on the first annealing portion of the firstoligonucleotide probe, wherein at the extension step the first quenchingoligonucleotide remains hybridized to the first tag portion, and (ii)cleavage and separation of the fluorescent moiety on the second tagportion from the third quenching moiety on the second annealing portionof the second oligonucleotide probe, wherein at the extension step thesecond quenching oligonucleotide remains hybridized to the second tagportion; (c) increasing temperature to a first temperature at which thefirst quenching oligonucleotide is not hybridized to the first tagportion from the first oligonucleotide probe and the second quenchingoligonucleotide remains hybridized to the second tag portion from thesecond oligonucleotide probe; (d) measuring a fluorescent signal at thefirst temperature; (e) increasing temperature to a second temperature atwhich the second quenching oligonucleotide is not hybridized to thesecond tag portion from the second oligonucleotide probe; (f) measuringa fluorescent signal at the second temperature; (g) obtaining acalculated signal value by subtracting the fluorescent signal detectedat the first temperature from the fluorescent signal detected at thesecond temperature; (h) repeating steps (b) through (g) in multiple PCRcycles to produce desired quantity of amplification products from thefirst and second target nucleic acid sequences; (I) determining thepresence of the first target nucleic acid sequence from the fluorescentsignals detected at the first temperature from the multiple PCR cyclesand the presence of the second target nucleic acid sequence from thecalculated signal values from the multiple PCR cycles.

In one embodiment, the first tag portion and the second tag portion bothcomprise a modification such that both tag portions are not capable ofbeing extended by a nucleic acid polymerase.

In one embodiment, the first tag portion is attached to the 3′ terminusof the first annealing portion of the first oligonucleotide probe andthe second tag portion is attached to the 3′ terminus of the secondannealing portion of the second oligonucleotide probe. In anotherembodiment, the first tag portion is attached to the 3′ terminus of thefirst annealing portion of the first oligonucleotide probe and thesecond tag portion is attached to the 5′ terminus of the secondannealing portion of the second oligonucleotide probe. In anotherembodiment, the first tag portion is attached to the 3′ terminus of thefirst annealing portion of the first oligonucleotide probe and thesecond tag portion is attached via a linker to a region of the secondannealing portion of the second oligonucleotide probe.

In one embodiment, the first tag portion is attached to the 5′ terminusof the first annealing portion of the first oligonucleotide probe andthe second tag portion is attached to the 5′ terminus of the secondannealing portion of the second oligonucleotide probe. In anotherembodiment, the first tag portion is attached to the 5′ terminus of thefirst annealing portion of the first oligonucleotide probe and thesecond tag portion is attached to the 3′ terminus of the secondannealing portion of the second oligonucleotide probe. In anotherembodiment, the first tag portion is attached to the 5′ terminus of thefirst annealing portion of the first oligonucleotide probe and thesecond tag portion is attached via a linker to a region of the secondannealing portion of the second oligonucleotide probe.

In one embodiment, the first tag portion is attached via a linker to aregion of the first annealing portion of the first oligonucleotide probeand the second tag portion is attached to the 5′ terminus of the secondannealing portion of the second oligonucleotide probe. In anotherembodiment, the first tag portion is attached via a linker to a regionof the first annealing portion of the first oligonucleotide probe andthe second tag portion is attached to the 3′ terminus of the secondannealing portion of the second oligonucleotide probe. In anotherembodiment, the first tag portion is attached via a linker to a regionof the first annealing portion of the first oligonucleotide probe andthe second tag portion is attached via a linker to a region of thesecond annealing portion of the second oligonucleotide probe.

In one embodiment, any of the first tag portion of the firstoligonucleotide probe or the first quenching oligonucleotide or thesecond tag portion of the second oligonucleotide probe or the secondquenching oligonucleotide or any combinations thereof contains one ormore nucleotide modifications. In one embodiment, the one or morenucleotide modifications is selected from the group consisting of LockedNucleic Acid (LNA), Peptide Nucleic Acid (PNA), Bridged Nucleic Acid(BNA), 2′-O alkyl substitution, L-enantiomeric nucleotide, orcombinations theoreof.

In yet another aspect, the invention provides for a kit for detectingtwo or more target nucleic acid sequences in a sample comprising: (a)two or more pairs of oligonucleotide primers with sequences that arecomplementary to each strand of the two or more target nucleic acidsequences; (b) at least one oligonucleotide probe comprising twodistinct portions, an annealing portion comprising a sequence at leastpartially complementary to one of the more than one target nucleic acidsequences and anneals within said one of the more than one targetnucleic acid sequences, wherein the annealing portion comprises a firstquencher moiety; and a tag portion attached to the 5′ terminus or to the3′ terminus of the first annealing portion or attached via a linker to aregion of the annealing portion, and comprising a nucleotide sequencethat is non-complementary to the more than one target nucleic acidsequences, wherein the tag portion comprises a fluorescent moiety whosedetectable signal is capable of being quenched by the first quenchermoiety on the annealing portion, wherein said fluorescent moiety isseparated from the first quenching moiety by a nuclease susceptiblecleavage site; (c) at least one quenching oligonucleotide comprising anucleotide sequence at least partially complementary to the tag portionof the oligonucleotide probe and hybridizes to the tag portion to form aduplex, wherein the quenching oligonucleotide comprises a secondquencher moiety which quenches the detectable signal generated by thefluorescent moiety on the tag portion when the quenching oligonucleotideis hybridized to the tag portion. In one embodiment, the tag portion ofthe oligonucleotide probe or the quenching oligonucleotide or both thetag portion of the oligonucleotide probe and the quenchingoligonucleotide contains one more nucleotide modifications wherein theone or more nucleotide modifications is selected from the groupconsisting of Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA),Bridged Nucleic Acid (BNA), 2′-O alkyl substitution, L-enantiomericnucleotide, or combinations theoreof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical description of one embodiment of theoligonucleotide probe used to perform the methods of the invention.

FIG. 2 is a graphical representation of the method of the invention thatshows the separation of the tag portion and subsequent dissociation ofthe quenching oligonucleotide.

FIG. 3 is a description of one embodiment of the methods of the presentinvention.

FIG. 4 shows the signal detection temperatures using another embodimentof the methods of the present invention.

FIG. 5 shows different embodiments of the oligonucleotide probes used topractice the methods of the present invention.

FIG. 6 shows the results of the hybridization and dissociation at twotemperatures between a quenching oligonucleotide and a fluorescentlylabeled complementary oligonucleotide as described in Example 1.

FIG. 7 shows the PCR growth curves generated from an internal controltemplate (GIC) at 0, 100, 1,000 or 10,000 cp/r×n using a standardTaqMan® probe G0 and FAM fluorescence readings at 58° C. and in theabsence of HIV-1 Group M template (HIM) (FIG. 7A) or in the presence ofHIM at 10 cp/r×n (FIG. 7B), 100 cp/r×n (FIG. 7C) and 1,000 cp/r×n (FIG.7D).

FIG. 8 shows the PCR growth curves generated from HIM at 0, 10, 100 or1,000 cp/r×n using a tagged probe (L24) with a complementary quenchingoligonucleotide (Q9) and FAM fluorescence readings at 80° C. and in theabsence of GIC (FIG. 8A) or in the presence of GIC at 100 cp/r×n (FIG.8B), 1,000 cp/r×n (FIG. 8C) and 10,000 cp/r×n (FIG. 8D).

FIG. 9 shows the derived growth curves from HIM at 0, 10, 100 or 1,000cp/r×n generated by having 84% of the 58° C. fluorescence signalssubtracted from the 80° C. fluorescence signals in the absence of GIC(FIG. 9A) or in the presence of GIC at 100 cp/r×n (FIG. 9B), 1,000cp/r×n (FIG. 9C) and 10,000 cp/r×n (FIG. 9D).

FIG. 10 shows the PCR growth curves generated from an internal controltemplate (GIC) or an HIV template (HIV), or both GIC and HIV templates(G+H) using a standard TaqMan® GIC probe (G0) and a tagged HIV probe(L24) with complementary quenching oligonucleotide (Q9) in which bothprobes are labeled with FAM (1st row), with HEX (2^(nd) with JA270 dye(3^(rd) row) or with Cy5.5 (4^(th) row).

FIG. 11 shows the PCR growth curves of the experiment as described inExample 4 in which the L24 tagged probe contains L-DNA instead of D-DNA.

FIG. 12 shows the PCR growth curves of the experiment as described inExample 5 in which fluorescence signal detection was measured at 58° C.,75° C., 88° C. or 97° C. in the presence of a standard TaqMan® GIC probe(IC-QF), a tagged HIV probe (L24) with a quenching oligonucleotide thathas A and G nucleotides modified with 2′-OMe substitutions (Q9-OMe A/G),a tagged HIV probe that has all nucleotides modified with 2′OMesubstitutions (L24-OMe) with a quenching oligonucleotide (Q9-OMe A/G),and a tagged HIV probe (L24-OMe) with a quenching oligonucleotide thathas all nucleotides modified with 2′-OMe substitutions (Q9-OMe).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “sample” as used herein includes a specimen or culture (e.g.,microbiological cultures) that includes nucleic acids. The term “sample”is also meant to include both biological and environmental samples. Asample may include a specimen of synthetic origin. Biological samplesinclude whole blood, serum, plasma, umbilical cord blood, chorionicvilli, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid(e.g., bronchioalveolar, gastric, peritoneal, ductal, ear,arthroscopic), biopsy sample, urine, feces, sputum, saliva, nasalmucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat,breast milk, breast fluid, embryonic cells and fetal cells. In apreferred embodiment, the biological sample is blood, and morepreferably plasma. As used herein, the term “blood” encompasses wholeblood or any fractions of blood, such as serum and plasma asconventionally defined. Blood plasma refers to the fraction of wholeblood resulting from centrifugation of blood treated withanticoagulants. Blood serum refers to the watery portion of fluidremaining after a blood sample has coagulated. Environmental samplesinclude environmental material such as surface matter, soil, water andindustrial samples, as well as samples obtained from food and dairyprocessing instruments, apparatus, equipment, utensils, disposable andnon-disposable items. These examples are not to be construed as limitingthe sample types applicable to the present invention.

The terms “target” or “target nucleic acid” as used herein are intendedto mean any molecule whose presence is to be detected or measured orwhose function, interactions or properties are to be studied. Therefore,a target includes essentially any molecule for which a detectable probe(e.g., oligonucleotide probe) or assay exists, or can be produced by oneskilled in the art. For example, a target may be a biomolecule, such asa nucleic acid molecule, a polypeptide, a lipid, or a carbohydrate,which is capable of binding with or otherwise coming in contact with adetectable probe (e.g., an antibody), wherein the detectable probe alsocomprises nucleic acids capable of being detected by methods of theinvention. As used herein, “detectable probe” refers to any molecule oragent capable of hybridizing or annealing to a target biomolecule ofinterest and allows for the specific detection of the target biomoleculeas described herein. In one aspect of the invention, the target is anucleic acid, and the detectable probe is an oligonucleotide. The terms“nucleic acid” and “nucleic acid molecule” may be used interchangeablythroughout the disclosure. The terms refer to oligonucleotides, oligos,polynucleotides, deoxyribonucleotide (DNA), genomic DNA, mitochondrialDNA (mtDNA), complementary DNA (cDNA), bacterial DNA, viral DNA, viralRNA, RNA, message RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA),siRNA, catalytic RNA, clones, plasmids, M13, P1, cosmid, bacteriaartificial chromosome (BAC), yeast artificial chromosome (YAC),amplified nucleic acid, amplicon, PCR product and other types ofamplified nucleic acid, RNA/DNA hybrids and polyamide nucleic acids(PNAs), all of which can be in either single- or double-stranded form,and unless otherwise limited, would encompass known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides and combinations and/or mixtures thereof. Thus, the term“nucleotides” refers to both naturally-occurring andmodified/nonnaturally-occurring nucleotides, including nucleoside tri,di, and monophosphates as well as monophosphate monomers present withinpolynucleic acid or oligonucleotide. A nucleotide may also be a ribo;2′-deoxy; 2′,3′-deoxy as well as a vast array of other nucleotide mimicsthat are well-known in the art. Mimics include chain-terminatingnucleotides, such as 3′-O-methyl, halogenated base or sugarsubstitutions; alternative sugar structures including nonsugar, alkylring structures; alternative bases including inosine; deaza-modified;chi, and psi, linker-modified; mass label-modified; phosphodiestermodifications or replacements including phosphorothioate,methylphosphonate, boranophosphate, amide, ester, ether; and a basic orcomplete internucleotide replacements, including cleavage linkages sucha photocleavable nitrophenyl moieties.

The presence or absence of a target can be measured quantitatively orqualitatively. Targets can come in a variety of different formsincluding, for example, simple or complex mixtures, or in substantiallypurified forms. For example, a target can be part of a sample thatcontains other components or can be the sole or major component of thesample. Therefore, a target can be a component of a whole cell ortissue, a cell or tissue extract, a fractionated lysate thereof or asubstantially purified molecule. Also a target can have either a knownor unknown sequence or structure.

The term “amplification reaction” refers to any in vitro means formultiplying the copies of a target sequence of nucleic acid.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification. Components of an amplificationreaction may include, but are not limited to, e.g., primers, apolynucleotide template, polymerase, nucleotides, dNTPs and the like.The term “amplifying” typically refers to an “exponential” increase intarget nucleic acid. However, “amplifying” as used herein can also referto linear increases in the numbers of a select target sequence ofnucleic acid, but is different than a one-time, single primer extensionstep.

“Polymerase chain reaction” or “PCR” refers to a method whereby aspecific segment or subsequence of a target double-stranded DNA, isamplified in a geometric progression. PCR is well known to those ofskill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; andPCR Protocols: A Guide to Methods and Applications, Innis et al., eds,1990.

“Oligonucleotide” as used herein refers to linear oligomers of naturalor modified nucleosidic monomers linked by phosphodiester bonds oranalogs thereof. Oligonucleotides include deoxyribonucleosides,ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs),and the like, capable of specifically binding to a target nucleic acid.Usually monomers are linked by phosphodiester bonds or analogs thereofto form oligonucleotides ranging in size from a few monomeric units,e.g., 3-4, to several tens of monomeric units, e.g., 40-60. Whenever anoligonucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′-3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, “T” denotes deoxythymidine,and “U” denotes the ribonucleoside, uridine, unless otherwise noted.Usually oligonucleotides comprise the four natural deoxynucleotides;however, they may also comprise ribonucleosides or non-naturalnucleotide analogs. Where an enzyme has specific oligonucleotide orpolynucleotide substrate requirements for activity, e.g., singlestranded DNA, RNA/DNA duplex, or the like, then selection of appropriatecomposition for the oligonucleotide or polynucleotide substrates is wellwithin the knowledge of one of ordinary skill.

As used herein “oligonucleotide primer”, or simply “primer”, refers to apolynucleotide sequence that hybridizes to a sequence on a targetnucleic acid template and facilitates the detection of anoligonucleotide probe. In amplification embodiments of the invention, anoligonucleotide primer serves as a point of initiation of nucleic acidsynthesis. In non-amplification embodiments, an oligonucleotide primermay be used to create a structure that is capable of being cleaved by acleavage agent. Primers can be of a variety of lengths and are oftenless than 50 nucleotides in length, for example 12-25 nucleotides, inlength. The length and sequences of primers for use in PCR can bedesigned based on principles known to those of skill in the art.

The term “oligonucleotide probe” as used herein refers to apolynucleotide sequence capable of hybridizing or annealing to a targetnucleic acid of interest and allows for the specific detection of thetarget nucleic acid.

A “reporter moiety” or “reporter molecule” is a molecule that confers adetectable signal. The detectable phenotype can be colorimetric,fluorescent or luminescent, for example. A “quencher moiety” or“quencher molecule” is a molecule that is able to quench the detectablesignal from the reporter moiety.

A “mismatched nucleotide” or a “mismatch” refers to a nucleotide that isnot complementary to the target sequence at that position or positions.An oligonucleotide probe may have at least one mismatch, but can alsohave 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides.

The term “polymorphism” as used herein refers to an allelic variant.Polymorphisms can include single nucleotide polymorphisms (SNP's) aswell as simple sequence length polymorphisms. A polymorphism can be dueto one or more nucleotide substitutions at one allele in comparison toanother allele or can be due to an insertion or deletion, duplication,inversion and other alterations known to the art.

The term “modification” as used herein refers to alterations of theoligonucleotide probe at the molecular level (e.g., base moiety, sugarmoiety or phosphate backbone). Nucleoside modifications include, but arenot limited to, the introduction of cleavage blockers or cleavageinducers, the introduction of minor groove binders, isotopic enrichment,isotopic depletion, the introduction of deuterium, and halogenmodifications. Nucleoside modifications may also include moieties thatincrease the stringency of hybridization or increase the meltingtemperature of the oligonucleotide probe. For example, a nucleotidemolecule may be modified with an extra bridge connecting the 2′ and 4′carbons resulting in locked nucleic acid (LNA) nucleotide that isresistant to cleavage by a nuclease (as described in Imanishi et al.,U.S. Pat. No. 6,268,490 and in Wengel et al., U.S. Pat. No. 6,794,499,both of which are incorporated herein by reference in their entireties).The compositions of the tag portion of the oligonucleotide probe and ofthe quenching oligonucleotide molecule are only restricted by theirability to form stable duplexes. These oligonucleotides can thereforecomprise of DNA, L-DNA, RNA, L-RNA, LNA, L-LNA, PNA (peptide nucleicacid, as described in Nielsen et al., U.S. Pat. No. 5,539,082), BNA(bridged nucleic acid, for example, 2′,4′-BNA(NC)[2′-O,4′-C-aminomethylene bridged nucleic acid] as described in Rahmanet al., J. Am. Chem. Soc. 2008; 130(14):4886-96), L-BNA etc. (where the“L-XXX” refers to the L-enantiomer of the sugar unit of the nucleicacids) or any other known variations and modifications on the nucleotidebases, sugars, or phosphodiester backbones.

Other examples of nucleoside modifications include various 2′substitutions such as halo, alkoxy and allyloxy groups that areintroduced in the sugar moiety of oligonucleotides. Evidence has beenpresented that 2′-substituted-2′-deoxyadenosine polynucleotides resembledouble-stranded RNA rather than DNA. Ikehara et al., (Nucleic AcidsRes., 1978, 5, 3315) have shown that a 2′-fluro substituent in poly A,poly I, or poly C duplexed to its complement is significantly morestable than the ribonucleotide or deoxyribonucleotide poly duplex asdetermined by standard melting assays. Inoue et al., (Nucleic AcidsRes., 1987, 15, 6131) have described the synthesis of mixedoligonucleotide sequences containing 2′-OMe (O-Methyl) substituents onevery nucleic nucleotide. The mixed 2′-OMe-substituted oligonucleotidehybridized to its RNA complement as strongly as the RNA-RNA duplex whichis significantly stronger than the same sequence RNA-DNA heteroduplex.Therefore, examples of substitutions at the 2′ position of the sugarinclude F, CN, CF₃, OCF₃, OMe, OCN, O-alkyl, S-alkyl, SMe, SO₂Me, ONO₂,NO₂, NH₃, NH₂, NH-alkyl, OCH₃═CH₂ and OCCH.

The term “specific” or “specificity” in reference to the binding of onemolecule to another molecule, such as a probe for a targetpolynucleotide, refers to the recognition, contact, and formation of astable complex between the two molecules, together with substantiallyless recognition, contact, or complex formation of that molecule withother molecules. As used herein, the term “anneal” refers to theformation of a stable complex between two molecules.

A probe is “capable of annealing” to a nucleic acid sequence if at leastone region of the probe shares substantial sequence identity with atleast one region of the complement of the nucleic acid sequence.“Substantial sequence identity” is a sequence identity of at least about80%, preferably at least about 85%, more preferably at least about 90%,95% or 99%, and most preferably 100%. For the purpose of determiningsequence identity of a DNA sequence and a RNA sequence, U and T oftenare considered the same nucleotide. For example, a probe comprising thesequence ATCAGC is capable of hybridizing to a target RNA sequencecomprising the sequence GCUGAU.

The term “cleavage agent” as used herein refers to any means that iscapable of cleaving an oligonucleotide probe to yield fragments,including but not limited to enzymes. For methods wherein amplificationdoes not occur, the cleavage agent may serve solely to cleave, degradeor otherwise separate the second portion of the oligonucleotide probe orfragments thereof. The cleavage agent may be an enzyme. The cleavageagent may be natural, synthetic, unmodified or modified.

For methods wherein amplification occurs, the cleavage agent ispreferably an enzyme that possesses synthetic (or polymerization)activity and nuclease activity. Such an enzyme is often a nucleic acidamplification enzyme. An example of a nucleic acid amplification enzymeis a nucleic acid polymerase enzyme such as Thermus aquaticus (Taq) DNApolymerase (TaqMan®) or E. coli DNA polymerase I. The enzyme may benaturally occurring, unmodified or modified.

A “nucleic acid polymerase” refers to an enzyme that catalyzes theincorporation of nucleotides into a nucleic acid. Exemplary nucleic acidpolymerases include DNA polymerases, RNA polymerases, terminaltransferases, reverse transcriptases, telomerases and the like.

A “thermostable DNA polymerase” refers to a DNA polymerase that isstable (i.e., resists breakdown or denaturation) and retains sufficientcatalytic activity when subjected to elevated temperatures for selectedperiods of time. For example, a thermostable DNA polymerase retainssufficient activity to effect subsequent primer extension reactions,when subjected to elevated temperatures for the time necessary todenature double-stranded nucleic acids. Heating conditions necessary fornucleic acid denaturation are well known in the art and are exemplifiedin U.S. Pat. Nos. 4,683,202 and 4,683,195. As used herein, athermostable polymerase is typically suitable for use in a temperaturecycling reaction such as the polymerase chain reaction (“PCR”). Theexamples of thermostable nucleic acid polymerases include Thermusaquaticus Taq DNA polymerase, Thermus sp. Z05 polymerase, Thermus flavuspolymerase, Thermotoga maritima polymerases, such as TMA-25 and TMA-30polymerases, Tth DNA polymerase, and the like.

A “modified” polymerase refers to a polymerase in which at least onemonomer differs from the reference sequence, such as a native orwild-type form of the polymerase or another modified form of thepolymerase. Exemplary modifications include monomer insertions,deletions, and substitutions. Modified polymerases also include chimericpolymerases that have identifiable component sequences (e.g., structuralor functional domains, etc.) derived from two or more parents. Alsoincluded within the definition of modified polymerases are thosecomprising chemical modifications of the reference sequence. Theexamples of modified polymerases include G46E E678G CS5 DNA polymerase,G46E L329A E678G CS5 DNA polymerase, G46E L329A D640G S671F CS5 DNApolymerase, G46E L329A D640G S671F E678G CS5 DNA polymerase, a G46EE678G CS6 DNA polymerase, Z05 DNA polymerase, ΔZ05 polymerase, ΔZ05-Goldpolymerase, ΔZ05R polymerase, E615G Taq DNA polymerase, E678G TMA-25polymerase, E678G TMA-30 polymerase, and the like.

The term “5′ to 3′ nuclease activity” or “5′-3′ nuclease activity”refers to an activity of a nucleic acid polymerase, typically associatedwith the nucleic acid strand synthesis, whereby nucleotides are removedfrom the 5′ end of nucleic acid strand, e.g., E. coli DNA polymerase Ihas this activity, whereas the Klenow fragment does not. Some enzymesthat have 5′ to 3′ nuclease activity are 5′ to 3′ exonucleases. Examplesof such 5′ to 3′ exonucleases include: Exonuclease from B. subtilis,Phosphodiesterase from spleen, Lambda exonuclease, Exonuclease II fromyeast, Exonuclease V from yeast, and Exonuclease from Neurospora crassa.

The term “propanediol” or “propanediol spacer” refers to 1,3-Propanedioland is synonymous with Propane-1,3-diol, 1,3-Dihydroxypropane, andTrimethylene glycol. The term “HEG” or “HEG spacer” refers tohexaethylene glycol, which is synonymous with3,6,9,12,15-Pentaoxaheptadecane-1,17-diol.

Various aspects of the present invention are based on a special propertyof nucleic acid polymerases. Nucleic acid polymerases can possessseveral activities, among them, a 5′ to 3′ nuclease activity whereby thenucleic acid polymerase can cleave mononucleotides or smalloligonucleotides from an oligonucleotide annealed to its larger,complementary polynucleotide. In order for cleavage to occurefficiently, an upstream oligonucleotide must also be annealed to thesame larger polynucleotide.

The detection of a target nucleic acid utilizing the 5′ to 3′ nucleaseactivity can be performed by a “TaqMan®” or “5′-nuclease assay”, asdescribed in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; andHolland et al., 1988, Proc. Natl. Acad. Sci. USA 88:7276-7280, allincorporated by reference herein. In the TaqMan® assay, labeleddetection probes that hybridize within the amplified region are presentduring the amplification reaction. The probes are modified so as toprevent the probes from acting as primers for DNA synthesis. Theamplification is performed using a DNA polymerase having 5′ to 3′exonuclease activity. During each synthesis step of the amplification,any probe which hybridizes to the target nucleic acid downstream fromthe primer being extended is degraded by the 5′ to 3′ exonucleaseactivity of the DNA polymerase. Thus, the synthesis of a new targetstrand also results in the degradation of a probe, and the accumulationof degradation product provides a measure of the synthesis of targetsequences.

Any method suitable for detecting degradation product can be used in a5′ nuclease assay. Often, the detection probe is labeled with twofluorescent dyes, one of which is capable of quenching the fluorescenceof the other dye. The dyes are attached to the probe, typically with thereporter or detector dye attached to the 5′ terminus and the quenchingdye attached to an internal site, such that quenching occurs when theprobe is in an unhybridized state and such that cleavage of the probe bythe 5′ to 3′ exonuclease activity of the DNA polymerase occurs inbetween the two dyes. Amplification results in cleavage of the probebetween the dyes with a concomitant elimination of quenching and anincrease in the fluorescence observable from the initially quenched dye.The accumulation of degradation product is monitored by measuring theincrease in reaction fluorescence. U.S. Pat. Nos. 5,491,063 and5,571,673, both incorporated by reference herein, describe alternativemethods for detecting the degradation of probe which occurs concomitantwith amplification.

A 5′ nuclease assay for the detection of a target nucleic acid canemploy any polymerase that has a 5′ to 3′ exonuclease activity. Thus, insome embodiments, the polymerases with 5′-nuclease activity arethermostable and thermoactive nucleic acid polymerases. Suchthermostable polymerases include, but are not limited to, native andrecombinant forms of polymerases from a variety of species of theeubacterial genera Thermus, Thermatoga, and Thermosipho, as well aschimeric forms thereof. For example, Thermus species polymerases thatcan be used in the methods of the invention include Thermus aquaticus(Taq) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermusspecies Z05 (Z05) DNA polymerase, Thermus species sps17 (sps17), andThermus species Z05 (e.g., described in U.S. Pat. Nos. 5,405,774;5,352,600; 5,079,352; 4,889,818; 5,466,591; 5,618,711; 5,674,738, and5,795,762. Thermatoga polymerases that can be used in the methods of theinvention include, for example, Thermatoga maritima DNA polymerase andThermatoga neapolitana DNA polymerase, while an example of a Thermosiphopolymerase that can be used is Thermosipho africanus DNA polymerase. Thesequences of Thermatoga maritima and Thermosipho africanus DNApolymerases are published in International Patent Application No.PCT/US91/07035 with Publication No. WO 92/06200. The sequence ofThermatoga neapolitana may be found in International Patent PublicationNo. WO 97/09451.

In the 5′ nuclease assay, the amplification detection is typicallyconcurrent with amplification (i.e., “real-time”). In some embodimentsthe amplification detection is quantitative, and the amplificationdetection is real-time. In some embodiments, the amplification detectionis qualitative (e.g., end-point detection of the presence or absence ofa target nucleic acid). In some embodiments, the amplification detectionis subsequent to amplification. In some embodiments, the amplificationdetection is qualitative, and the amplification detection is subsequentto amplification.

In the present invention, real-time PCR amplification and detection oftwo or more target nucleic acid in a single reaction vessel (e.g. tube,well) may be performed using a standard TaqMan® oligonucleotide probefor detecting the presence of the first target and one or more novelTaqMan® oligonucleotide probes for detecting the presence of the second,third or more targets, with all the probes containing the samefluorescent label. Alternatively, the novel TaqMan® oligonucleotideprobes may be used for detecting the presence of all the target nucleicacids. The novel probes have two distinguishing features.

The first feature of the novel probe is that it comprises two distinctportions. The first portion is referred as an annealing portion andcomprises a sequence that is at least partially complementary to atarget nucleic acid sequence such that it is capable of being hybridizedwith the target sequence. The annealing portion also contains a quenchermoiety. In one embodiment, the annealing portion contains a reportermoiety such as a fluorescent dye that is capable of being quenched thequencher moiety and is separated from the quencher moiety by a nucleasesusceptible cleavage site. The second portion of the oligonucleotideprobe is referred as a tag portion. In one embodiment, the tag portionis attached to the 5′ terminus of the annealing portion. In anotherembodiment, the tag portion is attached to the 3′ terminus of theannealing portion. In another embodiment, the tag portion is attachedanywhere between the 5′ terminus and the 3′ terminus of the annealingportion via a linker. The tag portion may comprise a nucleotide sequencethat is not complementary to the target nucleic acid sequence and formsa “flap” region that is not capable of binding to the target nucleicacid (see FIG. 1 for a graphical representation of a 5′ flap probe). Thetag portion may also be comprised of non-nucleotides such as any organicmoieties, or repeat units (e.g. (CH₂—CH₂—O)_(n), etc.) as long as it canbe attached to the annealing portion and can interact with a quenchingmolecule (as described in the following section). In one embodiment, thetag portion contains a reporter moiety such as a fluorescent dye that iscapable of being quenched by the quencher moiety on the annealingportion. The annealing and tag portions of the oligonucleotide probe mayoptionally be separated by a non-nucleotide “linker”. This linker can becomprised of carbon, carbon and oxygen, carbon and nitrogen, or anycombination of these and can be of any length. Furthermore, the linkercan be comprised of a linear moiety or a cyclic moiety. The linker maybe derived from a single unit or from multiple identical or differentunits separated by phosphate linkages. The purpose of the linker is tocreate a region at the junction of the annealing and tag portions of theoligonucleotide probe. When the tag portion is separated from theannealing portion, the linker may also prevent the tag portion frombeing extended by a nucleic acid polymerase. Alternatively, anothermodification on the separated tag portion renders it non-extendible bythe nucleic acid polymerase.

The second feature of the novel probe is that the tag portion binds to aquenching molecule. If the tag portion is a nucleotide sequence, thequenching molecule may be an oligonucleotide that is fully or partiallycomplementary to the nucleotide sequence of the tag portion andhybridizes to the tag portion. The quenching molecule also contains oris associated with a quencher moiety, i.e. a second quencher moiety,which is also capable of quenching the signal from the reporter moiety(e.g. fluorescent dye) on the tag portion. The quencher moiety on orassociated with the quenching molecule (the second quencher moiety) canbe the same as or different from the quencher moiety on the annealingportion (the first quencher moiety). Therefore, prior to performing PCRamplification, the reporter moiety on the tag portion is quenched byboth the quencher moiety on the annealing portion of the probe and bythe quencher moiety on or associated with the quenching molecule (e.g.by a quenching oligonucleotide as shown in FIG. 1).

The general principle of using the novel probe to perform real-time PCRamplification and detection of target nucleic acid in a 5′ nucleaseassay is described below. First, a sample suspected of containing thetarget nucleic acid is provided. The sample is then contacted inside asingle reaction vessel (e.g. a single test tube or a single well in amulti-well microplate) with PCR reagents that contain both theoligonucleotide primers capable of generating amplicons of the targetnucleic acid and the novel oligonucleotide probe. PCR amplificationbegins by using a nucleic acid polymerase having 5′ to 3′ nucleaseactivity such that during the extension step of each PCR cycle, thenuclease activity of the polymerase allows cleavage and separation ofthe tag portion from the quenching moiety on the annealing portion ofthe probe. The separated tag portion may optionally contain amodification (such as the non-nucleotide linker) such that it is notcapable of being extended by a nucleic acid polymerase.

Next, the signal from the reporter moiety on the separated tag portionis measured at a first temperature, usually, the annealing and/orextension temperature, at which the quenching molecule is still bound tothe tag portion. Due to the presence of the quencher moiety on orassociated with the quenching molecule, the signal from the reportermoiety (e.g. a fluorescent dye) on the tag portion is still quenched.Then, as a normal step in a PCR cycle, the temperature is graduallyraised to the denaturation temperature. As the temperature increasesfrom the extension temperature to the denaturation temperature, atemperature point is reached at which the quenching molecule is nolonger bound to the tag portion. If the quenching molecule is anoligonucleotide that has sequences complementary to the nucleotidesequence of the tag portion, this dissociation occurs at the meltingtemperature (Tm) of the duplex formation between the quenchingoligonucleotide molecule and the tag portion. Signal from the reportermoiety which is no longer quenched by the quencher moiety on orassociated with the quenching oligonucleotide is then measured at asecond temperature that is at or above the Tm temperature of the duplex.In fact, it may be better that the second temperature is above the Tmtemperature to ensure that close to 100% of the tag portion are insingle-stranded form. However, it is also possible to measure the signalat a temperature below the Tm temperature. Then, a calculated signalvalue is determined by subtracting the signal detected at the firsttemperature when the quenching molecule is still bound to the tagportion from the signal detected at the second temperature when thequenching molecule is not bound to the tag portion (see FIG. 2 and FIG.3). The calculated signal value may optionally be normalized forcorrection of signals that may be affected by temperature. For example,fluorescent signals are known to decrease at higher temperatures, andtherefore, standards can be used to normalize the signal values obtainedat different temperatures.

These signal measurements and calculations are performed at multiple PCRcycles and the determined cumulative signal values can be used todetermine not only the presence or absence but also the quantity of thetarget nucleic acid by determining the threshold value (Ct value) from aPCR growth curve generated from the signal values calculated plottedagainst PCR cycle number. In one embodiment, the signal measurements andcalculations are performed at each PCR cycle.

Multiplex PCR assays using only one reporter moiety (e.g. onefluorescent dye) is possible by designing oligonucleotide probes thathave tag portions hybridized to their respective quenchingoligonucleotide molecules at various Tm temperatures. For example,amplification and detection of three target nucleic acid in one reactioncan be achieved by using three oligonucleotide probes all labeled withthe same fluorophore. A standard TaqMan® oligonucleotide probe may beused to detect the first target by measuring the fluorescent signal at afirst temperature (usually the annealing temperature of a PCR cycle). Afirst “tagged” probe with a low Tm tag-quenching oligonucleotide duplexmay be used to detect the second target by measuring the calculatedfluorescent value at a second temperature at or above its Tm temperatureand that is higher than the first temperature. A second “tagged” probewith a high Tm tag-quenching oligonucleotide duplex may be used todetect the third target by measuring the calculated fluorescent value ata third temperature at or above its Tm temperature and that is higherthan the second temperature. (see FIG. 4) Theoretically, it would bepossible to use one TaqMan® probe and two different tagged probes withfour to seven different reporter moieties (e.g. fluorescent dyes) todetect between 12 and 21 different target nucleic acids in one reactionor one TaqMan® probe and 3 different tagged probes to detect between 16and 28 different target nucleic acids in one reaction.

Additionally, the novel probes of the present invention can be designedsuch that the tag portion is a nucleotide sequence and is connected to aquenching oligonucleotide to form a hairpin (i.e. a stem-loopstructure). In this structure, the “stem” portion will consist of thecomplementary regions between the tag portion and the quenchingoligonucleotide while the “loop” portion may be comprised ofnon-complementary nucleotides or non-nucleotides such as linkers aspreviously described.

Although the novel probes of the present invention have been describedas having the reporter moiety located on the tag portion of the probes,it is also possible to position the reporter moiety on the annealingportion and place the first quencher moiety on the tag portion, as longas the reporter moiety can reversibly interact with the second quenchermoiety on the quenching molecule. In a general sense, the reportermoiety is designed and positioned in the probe oligonucleotide in such away that it is separated from the first quenching moiety on theannealing portion during the 5′ nuclease (TaqMan®) assay and furtherdesigned to reversibly interact with the second quenching moiety on thequenching molecule. Some of these various alternate embodiments of thenovel probes can be seen in FIG. 5.

In order to practice the methods of the present invention, certainfeatures are necessary in the design of the tag portion of the probeoligonucleotide and of the quenching molecule. In one embodiment, boththe tag portion and the quenching molecule are comprised of nucleotidesequences. In this situation, both the tag portion and the quenchingoligonucleotide should not hybridize specifically to the target nucleicacid sequence but they should be fully or partially complementary toeach other to allow hybridization at the desired temperatures. Both mayinclude a modification at their 3′ termini in order to not be extendedby the nucleic acid polymerase during PCR amplification. Both thereporter moiety (e.g. fluorescent dye) on the tag portion and thequencher moiety on the quenching oligonucleotide can be located at the5′ terminus, the 3′ terminus or at any position between the 5′ and 3′termini but they must be located in proximity to each other when the tagportion is hybridized to the quenching oligonucleotide to allow thequenching moiety to quench the detectable signal from the reportermoiety.

With respect to different tag portions being hybridized to theirrespective quenching oligonucleotide molecules at various Tmtemperatures, modified nucleotides can be introduced at all or somepositions on either the tag portions, on the quenching oligonucleotidesor on both the tag portions and the quenching oligonucleotides such thatoligonucleotide length can be shortened. Examples of nucleotidemodifications that serve to increase the melting temperature includeLNA, PNA, G-clamp (9-(aminoethoxy)-phenoxazine-2′-deoxycytidine),propynyl deoxyuridine (pdU), propynyl deoxycytidine (pdC), and various2′ modifications at the sugar group, such as 2′-O-Methyl modifications.Another type of modification that may serve to prevent the unwantedbinding of nucleic acid polymerase to the tag portion or to thequenching oligonucleotide would include the use of enantiomeric L-formof a nucleotide, such as L-DNA, L-RNA or L-LNA.

In another embodiment, the tag portion of the oligonucleotide probe andthe quenching molecular are comprised of non-nucleotide molecules thatreversibly interact with each other in a temperature-dependent manner.Examples of such non-nucleotide interactions include but are not limitedto protein-protein interactions, protein-peptide interactions (e.g.peptide aptamers), protein-small molecule interactions, peptide-smallmolecule interactions, small molecule-small molecule interactions. Inone example, the well-known interaction between biotin and avidin (orstreptavidin) can be exploited by modifying either the biotin moiety(e.g. desthiobiotin) or the avidin moiety (see, Nordlund et al., J.Biol. Chem., 2003, 278 (4) 2479-2483) or both in order to make theinteraction reversible and temperature dependent.

In yet another embodiment, the interaction between the tag portion andthe quenching molecule may involve interaction between a nucleotidesequence (or nucleotide sequences) and a non-nucleotide molecule in asequence specific manner. Examples of these types of interactionsinclude but are not limited to nucleic acid aptamers, DNA bindingproteins or peptides and DNA minor groove binders. The design andsynthesis of sequence-specific DNA-binding molecules have been describedin several papers (see e.g. Dervan, Science, 1986, 232, 464-471; Whiteet al., Nature, 1998, 391, 468-471) and these methods may be used togenerate interactions between the tag portion and the quenching moleculethat are temperature-dependent. Similarly, interactions between doublestranded nucleotides and soluble quenchers can also be explored suchthat the quenching moiety does not need to be contained within thequenching molecule itself but may be in a soluble form that willinteract with and quench the reporter moiety only when the tag portionis bound to the quenching molecule. Embodiments of the present inventionwill be further described in the following examples, which do not limitthe scope of the invention described in the claims.

EXAMPLES Example 1 Verification of Quenching by the QuenchingOligonucleotide

An experiment was performed to verify that a quenching oligonucleotidecontaining a quencher moiety would be able to hybridize with thefluorescently-labeled tag portion of an oligonucleotide probe and quenchthe fluorescent signal at a temperature below the melting temperature ofthe duplex but not at a temperature above the melting temperature inwhich the duplex has been dissociated. Table 1 contains the nucleotidesequences of the tag portion and the quenching oligonucleotide.Quenching oligonucleotide Q0 does not contain the quencher whereasquenching oligonucleotide Q1 contains a BHQ-2 quencher at its 5′terminus.

TABLE 1 SEQ ID Name Sequence Modifications NO: 9FAM9TAGCGTCGCCAGTCAGCTCCGG9F9T 9 = C9 spacer,  1 F = FAM Q0CCGGAGCTGACTGGCGACGp p = phosphate 2 Q1 QCCGGAGCTGACTGGCGACGpp = phosphate,  3 Q = BHQ-2

The 9FAM9 TAG oligonucleotide was incubated without a quenchingoligonucleotide (QX) or with the Q0 or Q1 quenching oligonucleotide at1:5 molar ratio. The mixtures were then cycled in 50 μL reactions thatconsisted of 60 mM Tricine, 120 mM potassium acetate, 5.4% DMSO, 0.027%Sodium Azide, 3% glycerol, 0.02% Tween 20, 43.9 uM EDTA, 0.2 U/uL UNG,0.1 uM 19TAGC9FAMC9, 0.5 uM Q0 or Q1, 400 μM dATP, 400 μM4CTP, 400 μMdGTP, 800 μM dUTP, and 3.3 mM Manganese Acetate. Cycle conditionsresembling a typical PCR amplification reaction are shown on Table 2.

TABLE 2 Temperature Data Step Description Cycle # (° C.) Timeacquisition 1 Sterilization/RT  1 50  2 min none 94  5 sec none 55  2min none 60  6 min none 65  4 min none 2 Dark Cycles  5 95  5 sec none(no data 55 30 sec none acquisition) 3 TaqMan Cycles 55 91  5 sec none58 25 sec fluorescence read 80  5 sec fluorescence read

The results of the experiment are shown on FIG. 6. When the signal fromthe FAM dye was measured at 58° C., fluorescence was detected with noquenching oligonucleotide (QX) or with a quenching oligonucleotide withno quenching moiety (Q0) but no signal was detected at any of the cyclesin the presence of the Q1 quenching oligonucleotide. In contrast, whenfluorescence was measured at 80° C., signals could be detected in allcycles even in the presence of the Q1 quenching oligonucleotide, whichdemonstrates that at the higher temperature, the Q1 quenchingoligonucleotide was no longer hybridized with the TAG, and no quenchingwas observed.

Example 2 Real-Time PCR with Tagged Probe and Quenching Oligonucleotide

A Real-time PCR study was conducted using samples that contained variousconcentrations of an internal control template (GIC) mixed with variousconcentrations of a template sequence from HIV-1 Group M (HIM). Astandard TaqMan® hydrolysis probe (G0) that hybridizes to the GICsequence and a tagged probe (L24) with a complementary quenchingoligonucleotide (Q9) and an annealing portion that hybridizes to the HIMsequence were used to detect the amplification products generated fromthese two templates. Both probes were labeled with FAM and Table 3 showstheir sequences and the sequence of the quenching oligonucleotide.

TABLE 3 SEQ ID Name Sequence Modifications NO: G0FTGCGCGTCCCGQTTTTGATACTT F = FAM,  4 CGTAACGGTGCp Q = BHQ-2,p = phosphate L24 QTCTCTAGCAGTGGCGCCCGAACA F = FAM,  5 GGGACF Q = BHQ-2,CACACATTGGCACCGCCGTCTp p = phosphate,  tag underlined Q9AGACGGCGGTGCCAATGTGTGQp Q = BHQ-2,  6 p = phosphate

Four concentrations of GIC: 0 copies/reaction (cp/r×n), 100 cp/r×n,1,000 cp/r×n, and 10,000 cp/r×n were mixed with four concentrations ofHIM: 0 cp/r×n, 10 cp/r×n, 100 cp/r×n, and 1,000 cp/r×n to form sixteendifferent concentration combinations. PCR reagents and cycle conditionswere as described in Example 1 and Table 2 with the exception that 100nM of the G0 and L24 probes and 200 nM of the Q9 quenchingoligonucleotide were used in the reactions. Fluorescence readings fromthe FAM label were taken at 58° C. and at 80° C. for each cyclebeginning from cycle #6 (see Table 2).

The results of these experiments are shown on FIGS. 7-9. Thefluorescence readings at 58° C. are shown as growth curves in FIG. 7.FIG. 7A shows the growth curves generated with no HIM present and with0, 100, 1,000 or 10,000 cp/r×n GIC. Interestingly, there are essentiallyno differences in the fluorescence intensities and the Cycle threshold(Ct) values in the growth curve readings at 58° C. in the presence ofHIM at 10 cp/r×n (FIG. 7B), 100 cp/r×n (FIG. 7C) and 1,000 cp/r×n (FIG.7D) which indicate that only the FAM signal from the standard TaqMan® G0probe are detected at this temperature. This is because the FAM label onthe L24 tagged probe is very efficiently quenched by the quencher on theQ9 quenching oligonucleotide and does not interfere with the detectionof the GIC target.

The fluorescence readings at 80° C. are shown as growth curves in FIG.8. FIG. 8A shows the growth curves generated with no GIC present andwith 0, 10, 100 or 1,000 cp/r×n HIM. The fluorescence can now bedetected from the FAM label on the L24 probe because it is no longerquenched by both the quencher on the “annealing portion” of the probe(due to hydrolysis by the nuclease) and the quencher on the quenchingoligonucleotide (Q9) due to strand dissociation at this hightemperature. Although the fluorescence intensity from the L24 probe isconsiderably lower than that of the G0 probe, it is still sufficient tocalculate the Ct values that correspond to the starting concentrationsof HIM. However, when HIM and GIC are both present, the fluorescencereadings at 80° C. generate complex curves due to the strongerfluorescence that is detected from the G0 probe. (see FIG. 8B, 8C, 8D).Therefore, in order to “uncover” the fluorescent signal from the L24tagged probe, it would be necessary to subtract out the fluorescentsignal from the G0 probe, which would involve subtracting the 58° C.fluorescence readings (which is only contributed by the G0 probe) fromthe 80° C. fluorescence readings and derive growth curves that wouldresemble those observed in FIG. 8A.

When 100% of the 58° C. fluorescence readings were subtracted from the80° C. fluorescence readings, the derived growth curves showed negativevalues which indicated that there was overcompensation of thesubtraction. The reason for this observation was due to the reducedfluorescence intensity of the FAM label at 80° C. compared to theintensity at 58° C. Therefore, a “normalization” coefficient was deemednecessary and it was then empirically determined that 84% of the 58° C.signals subtracted from the 80° C. signals generated the best results.The derived growth curves are shown in FIGS. 9A, 9B, 9C and 9D and allare virtually identical to the 0 GIC growth curves of FIG. 8A. Theseresults show that that fluorescent signals that indicate the presence ofGIC can be separated from fluorescent signals that indicate the presenceof HIM and demonstrate the multiplexing utility of the presentinvention.

Example 3 Real-Time PCR with Probes Having Different Fluorescent Dyes

A series of experiments were performed as described in Example 2 exceptthat the G0 and L24 probes were labeled with FAM dye in the first set,with HEX dye in the second set, with JA270 dye in the third set and withCy5.5 dye in the fourth set. In each set of experiments, PCRamplification was performed with only GIC template present at 100cp/r×n, only HIV template present at 1000 cp/r×n or with both GIC (100cp/r×n) and HIV (1000 cp/r×n) templates present. The results of theexperiment are shown in FIG. 10. In fluorescence readings at 58° C.(FIG. 10, 1^(st) column), only signals generated by the G0 probes forthe GIC templates were observed, as expected, since the L24 probes werestill hybridized to the Q9 quenching oligonucleotides. In fluorescencereadings at 80° C. (FIG. 10, 2^(nd) column), signals generated by boththe G0 probes (for GIC) and the “unquenched” L24 probes for HIV) wereobserved. After using a normalized coefficient for each fluorescent dye,the 58° C. signals subtracted from the 80° C. signals generated thegrowth curves derived from the HIV template only (FIG. 10, 3^(rd)column). The signals generated from HEX and JA270 were similar or higherthan the signals from FAM while the signals from Cy5.5 were considerablylower than FAM signals but nevertheless detectable.

Example 4 Real-Time PCR with L-DNA Tagged Probe and QuenchingOligonucleotide

An experiment identical to the experiment described in Example 2 wasperformed with the exception that the L24 Tagged Probe to detect theHIV-1 Group M (HIM) template was comprised entirely of L-deoxyribosenucleotides instead of the “natural” D-deoxyribose nucleotides. Theresults of the experiment are shown in FIG. 11 where it was observedthat the fluorescence signals generated by using the L-enantiomer formof the L24 tagged probe were 4-5 fold higher than the signals generatedusing the D-enantiomer form of the L24 tagged probe.

Example 5 Real-Time PCR with Tagged Probes and QuenchingOligonucleotides Having 2′-O Methyl Modifications

An experiment similar to the one described in Example 2 was performedexcept that tagged probes and quenching oligonucleotides havingnucleotide modifications were used. In addition to the “standard” L24probe used to detect the presence of the HIM template, the tagged probe,L24-OME was generated in which every nucleotide in the tag portion ofthe probe (shown in TABLE 3 as the underlined portion of L24) wasmodified by having an O-Methyl substituent on the 2′ position of theribose moiety (2′-OMe). Two modified Q9 quenching oligonucleotides forhybridizing to the tag portion of L24 were also generated. Q9-OME hadevery nucleotide modified by a 2′-OMe substituent, and Q9-OME (A/G) hadonly the A and G nucleotides modified by a 2′-OMe substituent. Detectionof the HIM template was performed using three different combinations ofthe tag portion and quenching oligonucleotide: L24 with Q9-OME (A/G),L24-OME with Q9-OME (A/G) and L24-OME with Q9-OME. Results of thisexperiment are shown in FIG. 12.

As expected, at 58° C., only the fluorescent signal from the G0 TaqMan®probe could be detected. At 75° C., fluorescent signals were detectedfrom G0 and from L24/Q9-OME (A/G) but not from the two othertag-quenching oligonucleotide combinations. At 88° C., fluorescentsignals could also be detected from L24-OME/Q9-OME (A/G) and at 97° C.,signals were detected from all the probes, including the L24-OME/Q9-OMEcombination. These results show not only that fluorescent readings fromthree separate temperatures can be achieved using tagged probes andquenching molecules but that nucleotide modifications such as 2′-OMe canbe selectively introduced to the nucleotide sequence of the tag portionor to the quenching oligonucleotide or to both in order to alter themelting temperature of the tag-quenching oligonucleotide duplex withouthaving to change either their sequences or their lengths.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, the methods described above can be used invarious combinations. All publications, patents, patent applications,and/or other documents cited in this application are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent, patent application, and/or otherdocument were individually indicated to be incorporated by reference forall purposes.

INFORMAL SEQUENCE LISTING SEQ ID NO 1: 9FAM9TAG oligonucleotide sequenceCGTCGCCAGTCAGCTCCGGT SEQ ID NO 2: Q0 quenching oligonucleotidesequence (no quencher) CCGGAGCTGACTGGCGACGSEQ ID NO 3: Q1 quenching oligonucleotidesequence (BHQ-1 quencher on 5′ terminus) CCGGAGCTGACTGGCGACGSEQ ID NO 4: G0 TaqMan probe oligonucleotidesequence (FAM/BHQ/phosphate) TGCGCGTCCCGTTTTGATACTTCGTAACGGTGCSEQ ID NO 5: L24 Tagged probe oligonucleotidesequence (BHQ/FAM/phosphate)TCTCTAGCAGTGGCGCCCGAACAGGGACCACACATTGGCACCGCCGTCTSEQ ID NO 6: Q9 quenching oligonucleotidesequence (quencher on 3′ terminus) AGACGGCGGTGCCAATGTGTG

The invention claimed is:
 1. A kit for detecting two or more targetnucleic acid sequences in a sample by a real-time polymerase chainreaction (PCR) assay comprising: (a) two or more pairs ofoligonucleotide primers with sequences that are complementary to eachstrand of the two or more target nucleic acid sequences; (b) at leastone oligonucleotide probe comprising two distinct portions: (i) anannealing portion comprising a sequence at least partially complementaryto one of the two or more target nucleic acid sequences and annealswithin said one of the two or more target nucleic acid sequences,wherein the annealing portion comprises a first quencher moiety; and(ii) a tag portion attached to the 5′ terminus or to the 3′ terminus ofthe annealing portion or attached via a linker between the 5′ terminusand the 3′ terminus of the annealing portion, and comprising anucleotide sequence that is non-complementary to said one of the two ormore target nucleic acid sequences, wherein the tag portion comprises afluorescent moiety whose detectable signal is capable of being quenchedby the first quencher moiety on the annealing portion, wherein saidfluorescent moiety is separated from the first quenching moiety by anuclease susceptible cleavage site; (c) at least one quenchingoligonucleotide comprising a nucleotide sequence at least partiallycomplementary to the tag portion of the oligonucleotide probe andhybridizes to the tag portion to form a duplex, wherein said quenchingoligonucleotide comprises a second quencher moiety which quenches thedetectable signal generated by the fluorescent moiety on the tag portionwhen the quenching oligonucleotide is hybridized to the tag portion; and(d) a nucleic acid polymerase having 5′ to 3′ nuclease activity.
 2. Thekit of claim 1 wherein the tag portion is attached to the 5′ terminus ofthe annealing portion.
 3. The kit of claim 1 wherein the tag portion isattached via a linker between the 5′ terminus and the 3′ terminus of theannealing portion.
 4. The kit of claim 1 wherein the tag portion of theoligonucleotide probe or the quenching oligonucleotide or both the tagportion of the oligonucleotide probe and the quenching oligonucleotidecontains one or more nucleotide modifications.
 5. The kit of claim 4wherein the one or more nucleotide modifications comprises a nucleotidemodification selected from Locked Nucleic Acid (LNA), Peptide NucleicAcid (PNA), Bridged Nucleic Acid (BNA), 2′-O alkyl substitution,L-enantiomeric nucleotide, or combinations theoreof.
 6. The kit of claim5 wherein the nucleotide modification comprises LNA.
 7. The kit of claim5 wherein the nucleotide modification comprises PNA.
 8. The kit of claim5 wherein the nucleotide modification comprises BNA.
 9. The kit of claim5 wherein the nucleotide modification comprises L-enantiomericnucleotide.
 10. The kit of claim 9 wherein the nucleotide modificationcomprises L-enantiomeric LNA (L-LNA).
 11. The kit of claim 5 wherein thenucleotide modification comprises 2′-O alkyl substitution.
 12. The kitof claim 11 wherein the nucleotide modification comprises 2′-O methylsubstitution (2′-OMe).